1

Supplemental Materials and Methods

Generation of Recql4fl/fl mice

The targeting vector was generated using BAC clones from the C57BL/6J RPCI-23 BAC

library and was transfected into TaconicArtemis C57BL/6N Tac ES cell line. Gene targeting

was performed in the TaconicArtemis C57BL/6N Tac ES cell line. Exons 9 and 10 were

flanked by LoxP sites (size of loxP-flanked region: 1.0 kb) with a positive selection marker

(Puromycin resistance - PuroR) flanked by F3 sites inserted into intron 8. Homologous

recombinant clones were isolated using positive (PuroR) and negative (Thymidine kinase)

selection. Positive clones were selected and expanded and screened by Southern blot for

correct targeting events. A correctly targeted ES line was used to generate chimeras.

Chimerism was measured in chimeras (G0) by coat color contribution of ES cells to the

BALB/c host (black/white). Highly chimeric mice were bred to strain Flp-deleter C57BL/6

females (C57BL/6-Tg(CAG-Flpe)2 Arte) (1). Once Flp recombination was confirmed the Flpe

transgene was bred off the Recql4fl/fl line. Genotyping was performed by PCR using primers:

3303_41: CACTCTAGAAGAGGGAGTCAGATGG and 3303_42:

ACAGCAACAGAACAGCAACTACG to yield products of 165bp for the WT and 325bp for the

floxed allele. The deleted allele was detected by adding the primer

CGCGCGAAAGCTGAGGAGTT to primers 3303_41 and 3303_42 to generate a 256bp

product for the excised allele. All PCR products were sequence verified. We thank J Hartner

(TaconicArtemis, Cologne, Germany) for advice on targeting strategy.

Germline deletion of Recql4 was achieve by crossing Recql4fl/fl mice with TNAP-Cre (provided

by K Matthaei, Australian National University, Canberra, Australia (2)).

Histology

Tissue was collected and fixed immediately in 2-4% fresh paraformaldehyde.

Immunohistochemistry was performed on paraffin sections of tibiae. Antigen-retrieval was

undertaken using 10mM Citrate buffer pH 6.0 at 95C for 10 min. After endogenous

peroxidase and non-specific protein block [3% H2O2, 10% FCS, 10% Normal Horse Serum],

anti-cleaved caspase 3 mAb (0.65µg/ml, Cell Signaling) was incubated overnight at 4oC. After

secondary pAb (Polyclonal Swine Anti-Rabbit Immunoglobulins/Biotinylated Swine F(ab')2

Dako) and streptavidin horseradish peroxidase incubation (SA-HRP, Dako), tyramide signal

amplification (TSA, Perkin-Elmer) was used, followed again by SA-HRP incubation. Staining

was developed with DAB+ [3'3' Diamino benzidine, Sigma) and briefly counterstained with

Mayer's hematoxylin.

Immunoblot

Rabbit polyclonal antibodies against murine Recql4 were generated by GL Biochem

(Shanghai) Ltd. The antibody that showed specific activity against over-expressed Flag-

tagged mRecql4 was used for western blotting. This antibody was raised against a peptide

fragment spanning amino acids 74-93 (sequenced used QEPSCWGPHLSRAATQNTQS).

2

Western blots were performed on tumour material derived from Osx-Cre p53fl/flRecql4+/+ and

Osx-Cre p53fl/flRecql4fl/fl mice (to be described in detail elsewhere).

In vitro B and T cell assay after retroviral complementation LKS+ were isolated as previously described (3). Cells were cultured for 48 hours, infected as

described in retroviral complementation section and then after 48 hours placed into B or T cell

culture. For B or T cell cultures, 20,000 OP9 or OP9-DL1 cells/ml were plated in a 24-well

plate 48 hours before infected LKS+ were added and cultured as described (4).

Flow Cytometric Analysis of Intracellular γH2AX (Phospho-Histone H2A.XSer139) and

Cell Cycle

T and B cell precursors were collected from the co-cultures then fixed with 2%

paraformaldehyde for 20 mins at RT. Cells were permeabilized with ice-cold methanol on ice,

washed with PBS/BSA and resuspended in Alexa Fluor 647™ Rabbit anti-γH2AX antibody

clone 20E3 (Cell Signaling). Cell were washed, resuspended in PBS containing 20 µg/ml

Propidium Iodide (PI) and 100 µg/ml RNase A and analyzed by flow cytometry. Results were

analyzed with FlowJo software Version 9.0 (Treestar). γH2AX foci were visualized on a per

cell basis using a laser scanning cytometer (Compucyte).

Analysis of Recql4 expression in murine megakaryocytes

Raw CEL files downloaded from GEO (GSE6593) were processed in GenePattern using

ExpressionFileCreator v7 (method = RMA, quantile normalization = yes, background correct =

yes, compute present absent calls = yes, normalization method = median scaling). Probeset

1422922_at (RECQL4) was not detected above background in any sample.

Raw CEL files downloaded from GEO (GSE49664) were processed in GenePattern using

NormalizeAffymetrixST v 2.2 (algorithm = RMA, probesets = core, controls = include, chip =

Affymetrix_MoGene_1_0_st_v1.na33.2.mm9.transcript.chip). As P/M/A calls are not available

we define a gene to be detected if its expression is greater that the median of the control

probes, plus 1 s.d. Probeset 10430071 (Recql4) was detected in 3/4 HSC, 3/4 ERY and 0/4

MEG samples.

References: 1. Buchholz, F., Angrand, P.O., and Stewart, A.F. 1998. Improved properties of FLP

recombinase evolved by cycling mutagenesis. Nat Biotechnol 16:657-662. 2. Lomeli, H., Ramos-Mejia, V., Gertsenstein, M., Lobe, C.G., and Nagy, A. 2000.

Targeted insertion of Cre recombinase into the TNAP gene: excision in primordial germ cells. Genesis 26:116-117.

3. Walkley, C.R., Fero, M.L., Chien, W.M., Purton, L.E., and McArthur, G.A. 2005. Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells. Nat Cell Biol 7:172-178.

4. Smeets, M.F., Chan, A.C., Dagger, S., Bradley, C.K., Wei, A., and Izon, D.J. 2013. Fli-1 overexpression in hematopoietic progenitors deregulates T cell development and induces pre-T cell lymphoblastic leukaemia/lymphoma. PLoS One 8:e62346.

AnnexinV and 7AAD data from Day 14 post tamoxifen in indicated populations within the bone marrowAll animals fed tamoxifen containing chow for 14 daysControl = R26-CreERT2 ki/+ Recql4fl/+ (n=3); KO = R26-CreERT2 ki/+ Recql4fl/fl (n=4)Data expressed as mean +/- sem

Population Annexin V+/7AAD- Fold difference T test Annexin V+/7AAD+ Fold difference T test AnnexinV-/7AAD+ Fold difference T testControl Recql4fl/fl (KO/WT) Control Recql4fl/fl (KO/WT) Control Recql4fl/fl (KO/WT)

LT-HSC (LKS+CD150+CD105+) 21.0 +/- 4.4 13.8 +/- 0.77 0.65 0.114 21.5 +/- 2.8 21.1 +/- 4.35 0.98 0.946 9.06 +/- 3.9 19.2 +/- 3.0 1.99 0.104MPP (LKS+CD150-CD105-) 7.2 +/- 0.8 4.8 +/- 0.85 0.68 0.118 14.7 +/- 1.2 21.3 +/- 2.86 1.45 0.119 12.4 +/- 3.5 26.6 +/- 1.91 2.15 0.012

GMP 2.3 +/- 0.4 2.8 +/- 0.6 1.22 0.541 24.7 +/- 1.6 22.6 +/- 3.71 0.91 0.667 14.4 +/- 4.0 20.0 +/- 1.68 1.38 0.21MkP 4.7 +/- 0.3 4.4 +/- 0.87 0.95 0.842 24.9 +/- 0.6 25.2 +/- 3.56 1.01 0.942 20.0 +/- 4.9 29.4 +/- 2.4 1.47 0.084

Pre-MegE 5.6 +/- 0.9 4.7 +/- 0.89 0.84 0.519 11.9 +/- 2.6 16.1 +/- 3.23 1.35 0.383 5.5 +/- 1.7 18.2 +/- 2.44 3.31 0.011Pre-CFU-E 12.1 +/- 1.0 11.2 +/- 2.19 0.92 0.753 1.3 +/- 0.8 1.5 +/- 0.54 1.18 0.819 2.8 +/- 1.4 2.1 +/- 0.46 0.74 0.3

CFU-E 11.3 +/- 2.3 9.3 +/- 2.09 0.82 0.545 4.9 +/- 0.5 5.8 +/- 1.74 1.16 0.718 5.1 +/- 1.9 8.7 +/- 2.4 1.7 0.32

Granulocytes 5.3 +/- 0.7 7.6 +/- 0.77 1.42 0.92 1.9 +/- 0.3 3.4 +/- 0.32 1.79 0.023 2.1 +/- 0.3 3.1 +/- 0.49 1.49 0.172Monocytes/Macrophages 18.6 +/- 3.7 20.8 +/- 2.28 1.12 0.616 4.4 +/- 0.9 6.7 +/- 1.31 1.54 0.233 3.8 +/- 0.4 4.2 +/- 0.41 1.11 0.527

ErythroidCD71+ Ter119+ 5.6 +/- 0.9 6.6 +/- 1.6 1.19 0.436 1.71 +/- 0.1 3.0 +/- 0.38 1.74 0.039 2.4 +/- 0.7 2.6 +/- 0.56 1.1 0.783

CD71 mid Ter119+ 7.7 +/- 1.4 8.6 +/- 0.98 1.13 0.578 6.5 +/- 1.5 10.0 +/- 1.49 1.52 0.172 2.9 +/- 0.6 3.5 +/- 0.59 1.2 0.532CD71- Ter119+ 1.2 +/- 0.0 1.2 +/- 0.1 0.97 0.743 0.4 +/- 0.1 0.5 +/- 0.11 1.19 0.651 0.2 +/- 0.0 0.2 +/- 0.03 1.15 0.645

B lymphoidMature B (B220+IgM+) 22.6 +/- 2.8 18.4 +/- 1.96 0.81 0.258 16.7 +/- 3.5 21.2 +/- 1.24 1.27 0.226 1.9 +/- 0.4 2.1 +/- 0.54 1.14 0.733

PreProB 17.6 +/- 2.2 18.2 +/- 2.27 1.03 0.537 7.0 +/- 0.6 8.0 +/- 1.09 1.13 0.537 1.0 +/- 0.3 1.6 +/- 0.45 1.6 3.42ProB 4.89 +/- 0.1 5.7 +/- 0.89 1.18 0.441 1.4 +/- 0.1 3.3 +/- 0.49 2.4 0.022 1.0 +/- 0.2 2.0 +/- 0.52 2.09 0.165PreB 25.9 +/- 0.9 27.4 +/- 2.55 1.06 0.629 14.5 +/- 0.4 21.2 +/- 1.46 1.46 0.012 14.5 +/- 0.4 1.9 +/- 0.47 1.38 0.427

T lymphoidCD4+ CD8- 13.8 +/- 1.0 21.7 +/- 2.7 1.57 0.069 5.6 +/- 0.3 7.1 +/- 1.05 1.26 0.309 4.4 +/- 0.4 4.2 +/- 0.52 0.97 0.872CD4- CD8+ 6.2 +/- 0.5 7.7 +/- 0.56 1.25 0.147 15.5 +/- 2.2 24.9 +/- 3.96 1.61 0.148 9.8 +/- 0.6 10.6 +/- 0.63 1.08 0.495

Figure 5 Panel All statistical tests done with Ordinary One-Way ANOVA with multiple comparison test using Prism softwareJ Day 30 WBC count in PB Day 30 RBC count in PB

Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? SummaryRecql4fl/+ vs. Recql4fl/fl 4.493 2.373 to 6.613 Yes *** Recql4fl/+ vs. Recql4fl/fl 6.325 4.544 to 8.106 Yes ****Recql4fl/+ vs. p53fl/fl 0.8929 -1.227 to 3.013 No ns Recql4fl/+ vs. p53fl/fl -0.025 -1.806 to 1.756 No nsRecql4fl/+ vs. Recql4fl/fl p53fl/fl 3.943 1.823 to 6.063 Yes *** Recql4fl/+ vs. Recql4fl/fl p53fl/fl 4.725 2.944 to 6.506 Yes ****Recql4fl/fl vs. p53fl/fl -3.6 -5.992 to -1.208 Yes ** Recql4fl/fl vs. p53fl/fl -6.35 -8.359 to -4.341 Yes ****Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.55 -2.942 to 1.842 No ns Recql4fl/fl vs. Recql4fl/fl p53fl/fl -1.6 -3.609 to 0.4094 No nsp53fl/fl vs. Recql4fl/fl p53fl/fl 3.05 0.6584 to 5.442 Yes * p53fl/fl vs. Recql4fl/fl p53fl/fl 4.75 2.741 to 6.759 Yes ****

K BM cellularityTukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? SummaryRecql4fl/+ vs. Recql4fl/fl 9.208 5.738 to 12.68 Yes ****Recql4fl/+ vs. p53fl/fl -0.2167 -3.687 to 3.254 No nsRecql4fl/+ vs. Recql4fl/fl p53fl/fl 8.383 4.913 to 11.85 Yes ****Recql4fl/fl vs. p53fl/fl -9.425 -13.23 to -5.623 Yes ****Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.825 -4.627 to 2.977 No nsp53fl/fl vs. Recql4fl/fl p53fl/fl 8.6 4.798 to 12.40 Yes ****

L LT-HSC MPPTukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? SummaryRecql4fl/+ vs. Recql4fl/fl 0.0001833 -0.001116 to 0.001482 No ns Recql4fl/+ vs. Recql4fl/fl 0.002083 -0.01915 to 0.02332 No nsRecql4fl/+ vs. p53fl/fl 0.0007333 -0.0005656 to 0.002032 No ns Recql4fl/+ vs. p53fl/fl 0.003558 -0.01768 to 0.02480 No nsRecql4fl/+ vs. Recql4fl/fl p53fl/fl -0.0006417 -0.001941 to 0.0006573 No ns Recql4fl/+ vs. Recql4fl/fl p53fl/fl -0.01789 -0.03913 to 0.003345 No nsRecql4fl/fl vs. p53fl/fl 0.00055 -0.0008729 to 0.001973 No ns Recql4fl/fl vs. p53fl/fl 0.001475 -0.02179 to 0.02474 No nsRecql4fl/fl vs. Recql4fl/fl p53fl/fl -0.000825 -0.002248 to 0.0005979 No ns Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.01998 -0.04324 to 0.003289 No nsp53fl/fl vs. Recql4fl/fl p53fl/fl -0.001375 -0.002798 to 4.795e-005 No ns p53fl/fl vs. Recql4fl/fl p53fl/fl -0.02145 -0.04471 to 0.001814 No ns

M B220+IgM+ B cells B220+IgM- B cellsTukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? SummaryRecql4fl/+ vs. Recql4fl/fl 0.6949 0.2097 to 1.180 Yes ** Recql4fl/+ vs. Recql4fl/fl 1.414 0.2857 to 2.543 Yes *Recql4fl/+ vs. p53fl/fl 0.04517 -0.4400 to 0.5303 No ns Recql4fl/+ vs. p53fl/fl -0.3462 -1.475 to 0.7824 No nsRecql4fl/+ vs. Recql4fl/fl p53fl/fl 0.7489 0.3060 to 1.192 Yes ** Recql4fl/+ vs. Recql4fl/fl p53fl/fl 1.465 0.4352 to 2.496 Yes **Recql4fl/fl vs. p53fl/fl -0.6497 -1.210 to -0.08951 Yes * Recql4fl/fl vs. p53fl/fl -1.76 -3.064 to -0.4573 Yes **Recql4fl/fl vs. Recql4fl/fl p53fl/fl 0.05397 -0.4701 to 0.5780 No ns Recql4fl/fl vs. Recql4fl/fl p53fl/fl 0.05116 -1.168 to 1.270 No nsp53fl/fl vs. Recql4fl/fl p53fl/fl 0.7037 0.1797 to 1.228 Yes ** p53fl/fl vs. Recql4fl/fl p53fl/fl 1.812 0.5926 to 3.031 Yes **

N CD71+Ter119+ erythroid cells in BM CD71midTer119+ erythroid cells in BM CD71-Ter119+ erythroid cells in BMTukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? SummaryRecql4fl/+ vs. Recql4fl/fl 3.497 1.847 to 5.148 Yes *** Recql4fl/+ vs. Recql4fl/fl 0.007183 -0.6453 to 0.6597 No ns Recql4fl/+ vs. Recql4fl/fl 0.5287 -0.9528 to 2.010 No nsRecql4fl/+ vs. p53fl/fl 0.5843 -1.066 to 2.234 No ns Recql4fl/+ vs. p53fl/fl -0.5537 -1.206 to 0.09885 No ns Recql4fl/+ vs. p53fl/fl -2.275 -3.757 to -0.7936 Yes **Recql4fl/+ vs. Recql4fl/fl p53fl/fl 3.368 1.718 to 5.018 Yes *** Recql4fl/+ vs. Recql4fl/fl p53fl/fl 0.002358 -0.6502 to 0.6549 No ns Recql4fl/+ vs. Recql4fl/fl p53fl/fl 0.4507 -1.031 to 1.932 No nsRecql4fl/fl vs. p53fl/fl -2.913 -4.721 to -1.105 Yes ** Recql4fl/fl vs. p53fl/fl -0.5609 -1.276 to 0.1540 No ns Recql4fl/fl vs. p53fl/fl -2.804 -4.427 to -1.181 Yes ***Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.1294 -1.937 to 1.678 No ns Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.004825 -0.7196 to 0.7100 No ns Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.078 -1.701 to 1.545 No nsp53fl/fl vs. Recql4fl/fl p53fl/fl 2.784 0.9761 to 4.591 Yes ** p53fl/fl vs. Recql4fl/fl p53fl/fl 0.556 -0.1588 to 1.271 No ns p53fl/fl vs. Recql4fl/fl p53fl/fl 2.726 1.103 to 4.349 Yes **

O Thymus CellularityTukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? SummaryRecql4fl/+ vs. Recql4fl/fl 106.7 47.19 to 166.2 Yes ***Recql4fl/+ vs. p53fl/fl -8.775 -68.26 to 50.71 No nsRecql4fl/+ vs. Recql4fl/fl p53fl/fl 103.6 44.09 to 163.1 Yes **Recql4fl/fl vs. p53fl/fl -115.5 -178.2 to -52.75 Yes ***Recql4fl/fl vs. Recql4fl/fl p53fl/fl -3.1 -65.80 to 59.60 No nsp53fl/fl vs. Recql4fl/fl p53fl/fl 112.4 49.65 to 175.1 Yes ***

Thymic subsetsP CD4+ CD4+CD8+ CD8+

Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? SummaryRecql4fl/+ vs. Recql4fl/fl 8.461 -8.747 to 25.67 No ns Recql4fl/+ vs. Recql4fl/fl 91.66 48.37 to 134.9 Yes *** Recql4fl/+ vs. Recql4fl/fl 2.365 -15.15 to 19.88 No nsRecql4fl/+ vs. p53fl/fl -7.041 -24.25 to 10.17 No ns Recql4fl/+ vs. p53fl/fl 16.94 -26.34 to 60.22 No ns Recql4fl/+ vs. p53fl/fl -12.53 -30.05 to 4.983 No nsRecql4fl/+ vs. Recql4fl/fl p53fl/fl 7.298 -9.910 to 24.51 No ns Recql4fl/+ vs. Recql4fl/fl p53fl/fl 90.74 47.46 to 134.0 Yes *** Recql4fl/+ vs. Recql4fl/fl p53fl/fl 2.184 -15.33 to 19.70 No nsRecql4fl/fl vs. p53fl/fl -15.5 -33.64 to 2.638 No ns Recql4fl/fl vs. p53fl/fl -74.72 -120.3 to -29.09 Yes ** Recql4fl/fl vs. p53fl/fl -14.9 -33.36 to 3.566 No nsRecql4fl/fl vs. Recql4fl/fl p53fl/fl -1.163 -19.30 to 16.98 No ns Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.9165 -46.54 to 44.71 No ns Recql4fl/fl vs. Recql4fl/fl p53fl/fl -0.1805 -18.64 to 18.28 No nsp53fl/fl vs. Recql4fl/fl p53fl/fl 14.34 -3.801 to 32.48 No ns p53fl/fl vs. Recql4fl/fl p53fl/fl 73.8 28.18 to 119.4 Yes ** p53fl/fl vs. Recql4fl/fl p53fl/fl 14.72 -3.746 to 33.18 No ns

Symbol P value* 0.05** 0.01*** 0.001**** 0.0001ns not significant

1 2 3 9 1087654 11 12 13 14-15 16 17 18 19 20 21 22

ATP dependent DNA Helicase RecQ domain

Mfsd3Lrrc14

OsteosarcomaLymphoma

A. RECQL4 (human; Chromosome 8: 145,736,672-145,743,200 reverse strand)

Rothmund-Thomson Syndrome associated tumour mutations (RTS); adapted from Eur J Hum Genet. 2009 Feb;17(2):151-8

1 2 3 87654 11 12 13 14 15 16 17 18 19 20 21 22

Mfsd3

500 bp

C. Recql4 Recombined KO allele

Lrrc14

LoxP

Frameshift after recombination from exon 8Premature stop codon introduced in Exon 12

1 2 3 9 1087654 11 12 13 14 15 16 17 18 19 20 21 22

Mfsd3

500 bp

B. Recql4 Conditional Allele (C57BL/6-Recql4tm2272Arte)

Lrrc14

LoxP

LoxP

Frt

ATP dependent DNA Helicase RecQ domain

+ Cre

WT (165bp)

floxed (325bp)excised (256bp)

- + - +

Rosa26-CreERT2D. E.

Recql4 fl/+ Recql4 fl/flTamoxifen

Figure 1 Smeets et al

gDNA control (585bp) Recql4

fl/fl

Pan Actin (42kDa)

Recql4 (~140kDa)

Recql4

+/+

Kusa4

b10 (

wt)

Recql4

-3xFlag

(in K

usa4

b10)

-

Western Blot (tumor material)

Supplemental Figure 1. Outline of targeted allele and data from germ-line deleted allele analysisA. Human Recql4 locus with mutations in RTS patients indicatedB. Mouse Recql4 locus with inserted LoxP sites indicated. Exon 9 and 10 of mouse Recql4 are flanked by LoxP elementsC. Recombined Recql4 allele after Cre exposure. Frame shift predicted to introduce a stop codon in exon 12.D. Genomic PCR showing recombination of the floxed allele to produce the excised product of the expected size.E. Western blot of Recql4 expression in tumours from Cre+ Recql4+/+ and Cre+ Recql4fl/fl mice as indicated. Kusa4b10 endogenouscontrol and Recql4-3xFlag tagged expressing Kusa4b10 used as a control. Tumour model will be described elsewhere in detail.

Supplemental Figure 2

Supplemental Figure 2. Expression of Recql4 in murine hematopoietic cell subsets from Gene Expression Commons databaseThe expression of Recql4 in murine hematopoiesis based on data retrieved from the Gene Expression Commons database.Blue indicates low expression, red indicates highest expression.

Supplemental Figure 3

0.0

0.2

0.4

0.6

0.8

1.0

0

50

100

150

200

+/+ fl/flGenotype

Leuk

ocyt

es p

er s

plee

n (x

106 )

A.

0

1

2

3

4

Cel

ls p

er s

plee

n (x

106 )

+/+ fl/fl

Granulocytes

+/+ fl/fl

Macrophages

B.

0

10

20

30

40

C.

Cel

ls p

er s

plee

n (x

106 )

+/+ fl/fl

CD71+Ter119+

+/+ fl/fl

CD71mid

Ter119+

+/+ fl/fl

CD71-Ter119+

P<0.001

0

20

40

60

80

100

+/+ fl/fl

B220+IgM+

Cel

ls p

er s

plee

n (x

106 )

D.

Cel

ls p

er fe

mur

(x10

6 )

+/+ fl/fl

CD4+ T cells

+/+ fl/fl

CD8+ve T cells

P=0.05

0

5

10

15

20

25 P=0.01

Cel

ls p

er s

plee

n (x

106 )

+/+ fl/fl

CD4+ T cells

+/+ fl/fl

CD8+ve T cells

CD4

CD8

CD4+ve TCRβ

CD8+ve TCRβ

R26-CreERT2 ki/+

Recql4+/+

R26-CreERT2 ki/+

Recql4fl/fl

CD4

CD8

CD4+ve TCRβ

CD8+ve TCRβ

+/+ fl/fl

DN1

+/+ fl/fl

CD4+CD8+

+/+

CD8+

fl/fl +/+ fl/fl

DN2

+/+ fl/fl

DN3

+/+ fl/fl

DN4

10-1

100

101

102

103

P<0.01P<0.01

Cel

ls p

er th

ymus

(x10

6 )

0.00.10.20.30.40.5

2468

P=0.05

P<0.01

Cel

ls p

er th

ymus

(x10

6 )

+/+

CD4+

fl/fl

Spl

een

Spl

een

T ce

llsB

M T

cel

ls

Thym

us T

cel

ls

E.

F. G. H.

Supplemental Figure 3. Spleen, bone marrow T cell and thymus dataA. Spleen cellularity; B. Numbers of granulocytes and macrophages per spleen; C. Erythroid fractions based on CD71/Ter119 staining from the indicated genotype; D. B lymphocyte number per spleen .E. CD4 and CD8 mature T cells in the bone marrow and representative FACS plots of TCRβ staining on each population;F. T cells per spleen;G. Mature T cell subsets in the thymus;H. T cell progenitors in the thymus.Data expressed as mean ± sem with students’s T test statistic. *P<0.05, **P<0.001 or P value as indicated on the figure; n>5 per genotype.

10

100

1000

10000

+/+ fl/fl

CFU-GEMM

+/+ fl/fl

BFU-E

+/+ fl/fl+/+ fl/fl

CFU-M

+/+ fl/fl

CFU-G

+/+ fl/fl

CFU-GM

Col

onie

s pe

r fem

ur

Supp figure 4

P=0.028 P<0.001 P<0.001 P=0.10 P=<0.001

Supplemental Figure 4. Colony forming cell analysis per femurFrequency of each colony forming cell type per femur of the indicated genotypes.Data expressed as mean ± sem with students’s T test statistic. *P<0.05, **P<0.001 or P value as indicated on the figure; n>5 per genotype.

A.

Tamoxifen food30 days

Bone marrow transplant

R26-CreERT2 ki/+ Recql4+/+

R26-CreERT2 ki/+ Recql4fl/+

R26-CreERT2 ki/+ Recql4fl/fl

Congenic CD45.1

>20wk

0

5

10

15

20

25

7 weeks 15 weeks 24 weeks

Time post transplant

% M

ac-1

+ve

of C

D45

.2+v

e in

PB

fl/+fl/fl

+/+

0

20

40

60

80

100

% C

D45

.2+v

e in

PB

7 weeks 15 weeks 24 weeks

Time post transplant

fl/+fl/fl

+/+

BM CD45.2+

fl/flfl/+

+/Δ

con

trol

flΔWT

Supplementary Figure 5

Supplemental Figure 5. Profound selection against Recql4 deletion in hematopoietic stem cells.(A) Schematic of bone marrow transplant strategy. Whole bone marrow was isolated from donors of each genotype treated with tamoxifen for 30 days. Donor cells were competitively transplanted with CD45.1/CD45.2 competitor bone marrow into CD45.1 recipients. Peripheral blood total chimerism (CD45.2+ve) and myeloid chimerism (Mac-1+ve) are shown from one of two independent experiments. At end point CD45.2 cells were isolated from the bone marrow and genomic PCR performed to determine the status of Recql4; n=5 recipients per genotype per experiment; experiment performed twice.

R26-CreERT2 ki/+

Recql4+/+R26-CreERT2 ki/+

Recql4fl/fl

OP9

OP9-DL1

R26-CreERT2 ki/+

Recql4+/+R26-CreERT2 ki/+

Recql4fl/fl

OP9

OP9-DL1

A. B.

C.

Day 0 Day 7 Day 14 Day 210.1

1

10

100

1000 +/+fl/fl

OP9-DL1

Days in Culture

Fold

Incr

ease

Cel

l Num

ber

Day 0 Day 7 Day 14 Day 210.1

1

10

100

1000

OP9

Fold

Incr

ease

Cel

l Num

ber

Days in Culture

+/+fl/fl

Supplemental Figure 6. OP9 and OP9-DL1 culturesA. Representative phase contrast photos of hematopoietic cells of the indicated genotypes cultured on OP9 or OP9-DL1. Note thesignificantly larger cell size of the Recql4 deficient cultures.B. Fold change in cell numbers over the period of culture for cells cultured on OP9 and OP9-DL1 as indicatedC. Images of γH2A.X foci in cells from the indicated genotype/culture system.Scale bars as indicated.

Supplemental Figure 6

+/+ fl/fl

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

0

50

100

150Fold expansion on OP9-DL1

GFP- GFP+

+/+ fl/fl

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

0

200

400

600Fold expansion on OP9

GFP- GFP+

+/+ fl/fl

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

0

2 106

4 106

6 106

8 106Total cell number on OP9-DL1

GFP- GFP+

+/+ fl/fl

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

+/+ W

T

fl/fl W

T

+/+ K50

8A

fl/fl K

508A

0.0

5.0 106

1.0 107

1.5 107Total cell number on OP9

GFP- GFP+

A.

B.

C.

GFP

Control R26-CreERT2ki/+ Recql4+/+ R26-CreERT2ki/+ Recql4fl/fl

GFP-RECQL4 WT GFP-RECQL4K508A GFP-RECQL4 WT GFP-RECQL4K508A

ITD Day 7 Day 14 Day 210

1

2

3

4

5

Fold

cha

nge

in G

FP

OP9 cultures - WT RECQL4

ITD Day 7 Day 14 Day 210

1

2

3

4

5

Fold

cha

nge

in G

FP

OP9 cultures RECQL4K508A

Supplemental Figure 7

Supplemental Figure 7. Retroviral complementation additional informationA. Representative FACS plots showing GFP expression in virally transduced cellsB. Fold expansion of cells of the indicated genotype on OP9-DL1 (T cell cultures) for GFP- (non rescued) or GFP+ (rescued)C. Fold expansion of cells of the indicated genotype on OP9 (B cell cultures) for GFP- (non rescued) or GFP+ (rescued)D. Fold change in GFP levels in OP9 cultures (B cells) for the EGFP-RECQL4 or EGFP-RECQL4K508A

D.